Anti-idiotypic monoclonal antibody reactive with HIV neutralizing antibody 2F5

ABSTRACT

The invention relates to Ab2-type anti-idiotypic antibodies and fragments thereof which mimic HVI-1 epitopes that are otherwise cryptic to the immune system and which antibodies or fragments thereof are directed against potently neutralizing anti-HIV-1 antibodies. The invention further relates to a hybridoma cell line 3H6 expressing the anti-idiotypic antibody and to pharmaceutical compositions containing the antibody or fragment thereof. The invention also relates to HIV-1 neutralizing Ab3-type antibodies elicited upon administration of the Ab2-type anti-idiotypic antibody or fragment thereof and to pharmaceutical compositions containing them. The invention also relates to the use of the present antibodies or fragments thereof as screening tools or as diagnostic or therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Phase of International Application No.PCT/EP03/00455, filed Jan. 17, 2003.

TECHNICAL FIELD

The present invention is in the fields of immunology and vaccinedevelopment and relates to antibodies that mimic an epitope on gp41 ofHIV-1. The invention further relates to applications of the antibodiesand fragments thereof, including their use for eliciting HIV-1neutralizing antibodies in mammalian hosts and to HIV-1 neutralizingantibodies elicited by said anibodies or antibody fragments. Theinvention also relates to pharmaceutical compositions, in particularvaccines, containing such antibodies or antibody fragments for eitheractive or passive immunization to inhibit or prevent HIV-1 infection inmammalian individuals.

BACKGROUND OF THE INVENTION

Anti-idiotypic antibodies (Ab2) are directed against the antigen bindingsite of other antibodies (Ab1). Epitopes of diverse pathogenic antigensmay be mimicked functionally and structurally by those Ab2s. Thus, Ab2sare able to induce anti-anti-idiotypic antibodies (Ab3) that areAb1-like and have similar biological properties [1].

Different kinds of Ab2s are distinguished after immunization with Ab1[2, 3]. The Ab2α (Ab2-alpha) recognizes idiotypes at the distal positionfrom Ab1 paratope and does not interfere with Ab1 binding to the nominalepitope. Ab2s beta (Ab2β) are directed to the paratope of Ab1s and bearthe internal image of the antigen. Ab2β can compete with the nominalantigen for binding to the idiotype and to antigen-specific xenogeneicantibodies. A third category, termed Ab2γ (Ab2-gamma) recognizesidiotopes physically close to the binding site but does not behave as asurrogate antigen [4].

Moreover, internal image anti-idiotype antibodies may exhibit identicalthree-dimensional conformation or amino-acid sequence homology with theepitope. This has been shown for the reovirus system, where the aminoacid sequence of the idiotype had the same motif as the virushemagglutinin [5]. Thus the virus-receptor binding on susceptible cellscan be prevented.

Generally, the advantages of anti-idiotypic antibodies over othervaccines applying peptide antigens or inactivated viruses can bemanifold [6]. They do not involve any viral components, they are safebut do not miss any specificity by their mimicry of single epitopes.They can be produced easily in large quantities for immunization. Ab2scan even induce an immune response against single neutralizing epitopesthat may not be immunogenic on the native antigen. They thus can breakimmunogenic unresponsiveness to certain antigens as has beendemonstrated in neonatal mice [7]. Active immunotherapy with Ab2-basedvaccines have proven very potent to induce antigen-specific Ab3 incancer patients against tumor cells [8-11].

Ab2s that display internal images of antigen (Ab2β) have also inducedprotective immunity against infective pathogens [12].

HIV-1 infections may be accessible to Ab2 therapy because of theimportance of neutralizing antibodies in the control of HIV-1 infections[13]. The possibility to induce neutralizing Ab3 in various systemsincluding HIV-1 has been described [14].

Cross-reactivity of HIV-1 antigens with various normal human antigenssuggests a potential advantage of single epitope Ab2 vaccines overmultiple epitope vaccines [15]. Different approaches for the inductionof a neutralizing humoral immune response have been made withanti-idiotypic antibodies. The most promising experiments wereundertaken with anti-CD4 specific mAbs, which are able to induce HIV-1neutralizing antibodies in vitro [16, 17]. The anti-CD4 mAbs Leu3a andIOT4A were also tested in clinical studies [18, 19] and were-shown toinduce a gp120 cross-reacting immune response that inhibits gp120binding to CD4. Ab2s against V3 loop peptides were also generated andused in different in vitro studies [20-22]. In different animal modelsthey induced Ab3s with the ability of binding the autologous peptides aswell as gp120, but could not neutralize HIV-1 in an in vitro experiment.Only one Ab2 has been described having the potential as a vaccineagainst HIV-1 [23].

In principle, Ab2 beta antibodies raised against antibodies neutralizingHIV-1 might have an enormous potential for vaccine design. A vaccineagainst HIV-1 must induce both, cellular as well as humoral immuneresponses. HIV-1 specific CTL-responses can be induced by variousexperimental vaccines such as DNA-vaccines [24] or chimeric influenzalive viruses administered as nasal spray and expressing HIV-1 epitopes[25]. Reasonable experimental vaccines inducing neutralizing antibodyresponses are so far generally not available. The human monoclonalantibody 2F5 (produced by hybridoma cell line ECACC Accession No.90091704) broadly and potently neutralizes primary HIV-1 isolates [26].Thus, mAb 2F5 is an extraordinary interesting tool for the design of ananti-HIV-1 vaccine leading to properly induced humoral immune responses.It recognizes the highly conserved six amino acid core epitope sequenceELDKWA (SEQ ID NO:1) on the virus envelope ectodomain gp41 [27].2F5-like specific antibodies are only rarely found in sera of HIV-1infected individuals. HIVIG (pooled sera of more than 70 HIV-1 positivedonators) do not show significant 2F5-like specific binding to gp160and/or gp41. The region on gp41 to which 2F5 binds is obviously crypticto the human immune system during natural infection. Differentmechanisms might contribute to its immuno-cryptic behavior. Oneexplanation might be that the human complement factor H which isabundantly present in normal human serum has a binding domain in thatregion of gp41 thus masking it against immune recognition [28]. ThismAb, although being a broad and potent entry blocker (neutralizers) ofHIV-1 binds only very weakly to free virus and HIV-1 infected cells.[30]. It is therefore concluded that this fusogenic region on gp41 isonly exposed during the event of fusion of the HIV-1 with the host cell.That might on the one hand give an additional explanation for thecryptic nature of these neutralizing epitopes during natural infection.On the other hand HIV-1 isolates that are neutralization insensitive tothis antibody have not been found so far.

In earlier studies the core epitope motif ELDKWA (SEQ. ID NO: 1) of 2F5was integrated into different antigenic formats. The presentation of theELDKWA-motif (SEQ ID NO: 1) on the haemagglutinin of influenza lifevirus was able to induce long lasting mucosal immune responses detectedas 2F5-like specific IgA's in mouse faeces upon repeated immunization asa mucosal nasal spray [25]. However, quantities of IgA sufficient toprove in vitro neutralization potency could not be extracted from thefaeces samples. Other forms of presenting the 2F5-core epitope forimmunization such as fusion to the hepatitis B surface antigen expressedin yeast induced very high 2F5-like specific ELISA-titers in immunizedBALB/c mice. However, the sera of those animals did not show anysignificant in vitro neutralization potency [36]. Peptide versions ofthe 2F5 epitope were also poorly immunogenic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reduction of binding capacity of 2F5 to the precoatedepitope after preincubation of 2F5 with Ab2 3H6. Serial dilutions of 2F5starting with 100 ng/mL were preincubated with 50 ng/mL or 500 ng/mL 3H6or with the unspecific Ab2 6H8. After 1 hour the dilutions were allowedto react with GGGLELDKWASL (SEQ ID NO:13) at the microtiter plate and2F5 was detected.

FIG. 2 shows a neutralization assay of 10 μg/mL 2F5 and 2G12 withincreasing amounts of 3H6 and irrelevant mouse IgG.

FIG. 3 shows the binding of ascites and serum samples of 3H6 immunizedmice to gp160.

FIG. 4 shows the binding of ascites and serum samples of 3H6 immunizedmice to GGGLELDKWASL (SEQ ID NO.13).

FIG. 5 shows the result of a competition assay of 3H6 immunized micesera with 2F5 in an ELISA binding study to precoated GGGLELDKWASL (SEQID NO:13).

FIG. 6 is a schematic delineation of the fusion protein mo/hu3H6/IL-15.

DESCRIPTION OF THE INVENTION

It is an object of the invention to generate antigen binding componentswhich are capable to induce neutralizing humoral immune responsesagainst HIV-1. A further object of the invention is to provide novelantibodies or antibody fragments which are able to induce HIV-1neutralizing antibodies.

The invention discloses murine antibodies and fragments thereof whichare directed against an antibody of the gp41 antigen on HIV-1. Inparticular, the invention describes the generation of anti-idiotypemurine Ab2β directed against the human monoclonal antibody 2F5 which isknown to react with a cryptic neutralizing epitope on gp41 of HIV-1. Oneof the Ab2's of the present invention, i.e. the murine monoclonal Ab2designated 3H6 (deposited under the provisions of the Budapest Treaty onthe Deposit of Microorganisms at PHLS Porton Down, Salisbury, UK; ECACCAcc. No. 01100279) blocks the binding of the human Ab1 2F5 to asynthetic epitope containing at least the core epitope sequence ELDKWA(SEQ ID NO:1) as well as to gp160. 3H6 is also able to diminish theHIV-1 neutralizing potency of 2F5.

The present invention also relates to Fab-fragments derived from 3H6antibodies, which were shown to induce neutralizing immune responses inthe sera of human and non-human mammals applying a simple prime/boostregimen of immunization. Thus, 3H6 Fab-fragments successfully mimick anepitope that apparently lacks immunogenicity or is only poorlyimmunogenic on the native HIV-1 during natural infection.

An “antibody fragment” in the context of the present invention is anantigen-binding part of the whole antibody which retains the bindingspecificity of the whole antibody molecule. The term encompasses, forexample, Fab-, F(ab′)2-, Fv- and scFv-fragments. Antibody fragments canbe obtained by conventional techniques well known in the art such asproteolytic digestion of antibodies, for example by papain digest, orthrough genetic engineering.

The antibodies and antibody fragments of the present invention can alsobe prepared by synthetic methods known in the art, such as peptidesynthesis or recombinant DNA technology, once the structural nature ofthe antibody fragments has been analyzed by crystallization studies,sequencing methods and/or other methods well known in the art.

In another embodiment, the invention relates to “humanized” antibodiesand antibody fragments. “Humanized” means that a non-human antibody orantibody fragment is linked with part of a human antibody. Usually itmeans that the complimentarity determining regions (CDR) from thevariable regions of a non-human, e.g. rodent, antibody are combined withframework regions from the variable regions of a human antibody, e.g.from a human IgG. In doing so the fragment containing the antigenbinding site of the non-human antibody is inserted into a suitable humanantibody. Methods for “humanizing” antibodies are state of the art. Forinstance, an exon encoding the variable region containing the CDR of thenon-human antibody may be spliced into the framework sequence of asuitable human antibody. Further, a Fab-fragment of the non-humanantibody may be coupled with a suitable Fc-fragment of a suitable humanantibody. Suitable human frameworks may be found by homology searches indatabases on the basis of homology to the non-human antibody.

“Humanized” antibody fragments according to the present invention mayalso be derived from synthetic phage libraries which use randomizedcombinations of synthetic human antibody variable regions, by selectingon 2F5 antibodies as antigen. Humanized antibodies are referred tohereinafter by using the prefix “hu”, e.g. the term “hu3H6” refers to ahumanized 3H6 antibody.

In a still further embodiment the invention relates to chimericantibodies. “Chimeric” antibodies are antibodies in which the whole ofthe variable regions of a non-human, in particular rodent, antibody areexpressed along with human constant regions of antibodies. This mayprovide antibodies with human effector functions. “Chimeric” antibodiesmay be created on the molecular level by fusing non-human exons encodingvariable regions containing the antigen binding site with human exonsencoding antibody constant regions. Chimeric mouse/human antibodies areidentified hereinafter by the prefix “mo/hu”, e.g. the term “mo/hu3H6”refers to a chimeric mouse/human 2H6 antibody.

Where the term “3H6” is used without identifying prefix the term shallrelate to the murine Ab2 antibody 3H6, e.g. as produced by cell line 3H6(ECACC Acc. No. 01100279), unless another meaning is derivable from therespective context. Such other meaning would include using the term 3H6as a generic term encompassing each one or all of the group elements:murine, chimeric and humanized 3H6 antibodies.

In yet another embodiment the present invention relates to anantibody-cytokine construct, wherein the vH and vL chains of a chimericmo/hu3H6 antibody (FIG. 6) are fused with human Fc and ckappa chains,resulting in a recombinant expression protein having an IgG isotype. Achimeric IgG molecule is able to pass the placenta, wherefore it isexpected that the aforementioned construct may sucessfully interferewith and inhibit or prevent a mother-to-child transmission of HIV-1.

Anti-idiotypic antibody 3H6 Fab-fragments as well as the mo/hu3H6Fc-gamma antibodies induce humoral immune responses and trigger theimmune system to induce neutralising antibodies.

In a further embodiment the present invention relates to an antibody orantibody fragment that is associated, e.g. fused or coupled, with animmunoactive molecule that improves the immunogenic nature of theantibodies induced by the antibody or antibody fragment uponadministration to a mammalian individual. For instance, the antibodyfragment may be coupled to a structure, for instance a cytokine such as,e.g., IL-4, which enhances the immunoreactivity of said mammal andpreferably causes an increased B-cell response, which is of particularimportance in the treatment of HIV. The present invention also relatesto entire Ab2 antibodies, particularly to antibody 3H6 including murine3H6, chimeric mo/hu3H6 and hu3H6, wherein the antibody is associated(e.g. coupled, fused or linked) with any such immunoresponse stimulatingmolecule, as well as to mixtures comprising at least one antibody and/oranti-HIV-effective fragment thereof in combination with at least onecytokine-associated, particularly IL-4 associated, antibody and/oranti-HIV-effective fragment thereof. The term “anti-HIV-effectivefragment” as used herein relates to antibody fragments that are reactivewith HIV-1 neutralizing antibody 2F5 (ECACC Accession No. 90091704) andwhich inhibit or prevent the HIV-1 neutralization activity of antibody2F5 and/or the binding of antibody 2F5 to gp41 of HIV-1, and which arepreferably effective in eliciting, upon proper administration to amammal, 2F5 type anti-HIV-1 antibodies.

Therapeutic use of interleukines in the treatment of cancer and viralinfections is applied since years. Additionally, some cytokines likeIL-2 are diminished in early phases of infection. Especially in HIV-1therapies considerable indications and independent studies point outthat IL-15 may be of great benefit.

Similarly to IL-2, IL-15 is responsible for the proliferation ofactivated T- and NK-cells, assists the distribution of IFN-γ andstimulates the generation of immune-globulines in B-cells. IL-15 mRNA isdetected in different tissues and is regulated tightly. IL-15 inducesproliferation of CD8⁺ memory cells, while IL-2 inhibits theproliferation of CD8⁺ memory cells. Therefore, IL-15 was chosen as apossible candidate in the concept of developing an improved HIV vaccineaccording to the present invention, and particularly at least for thereasons that it a) supports the generation of neutralising antibodies ina patient's B lymphocytes, and b) also stimulates the cellular immunityby increasing the number of CD8⁺ memory cells of said patient. Moreover,while infusion of IL-2 to AIDS patients showed severe side effects athigher doses, IL-15 is far less harmful and is even detected in new-bornbabies.

Accordingly, in a further embodiment the present invention relates to ananti-idiotypic Ab2 antibody or antibody fragment reactive with antibody2F5, preferably to Ab2 3H6 or a fragment thereof, that is associated,e.g. fused or coupled, with IL-15 as an immunoactive molecule. In apreferred embodiment, the invention relates to a fusion proteinconsisting of the chimeric mo/hu3H6 or humanized antibody hu3H6 andhuman IL-15, i.e. to a recombinant protein which is a fusion proteinconsisting of mo/hu3h6 or hu3H6 and a human IL-15 tail at the C-terminusof the antibody heavy chain (FIG. 6). The fusion proteins induceneutralising, 2F5 like antibodies in a mammalian recipient while at thesame time proliferation of CD8⁺ memory cells is stimulated through theaction of the IL-15 tail.

Moreover, apart from the aforementioned activity of thecytokine-associated Ab2 antibodies of the present invention to inducememory T cell activity, a combination of an Ab2 antibody oranti-HIV-effective fragment thereof and a cytokine-associated Ab2antibody or anti-HIV-effective fragment thereof allows to developoptimal therapeutic effects by adjusting the ratio of the components toeach other, typically in an anti-HIV-1 vaccine. In a preferredembodiment, the invention relates to a mixture or composition, typicallyan HIV-1 vaccine, comprising at least one antibody or effective fragmentthereof selected from the group consisting of 3H6, fragment of 3H6,mo/hu3H6, fragment of mo/hu3H6, hu3H6, and fragment of hu3H6 incombination with at least one cytokine-associated, preferably IL-4 orIL-15 associated antibody or effective fragment thereof, the antibody orfragment being selected from the same group consisting of 3H6, fragmentof 3H6, mo/hu3H6, fragment of mo/hu3H6, hu3H6, and fragment of hu3H6.

The proper ratio can be determined using in vitro models for eachrecipient prior to administration. Usally this determination willcomprise decreasing the Ab2-associated IL-15 concentration to a minimallevel and to supplement the hu3H6 antibody up to a concentration thatensures at least some, preferably the best possible induction of thehumoral immune response.

Sera of 3H6 induced immune responses show both specific bindinginhibition patterns of 2F5 on the antigen as well as significant invitro neutralization potency. Furthermore, the anti-2F5-Ab2β 3H6, whilereacting strongly with the homologous Ab1, does not recognize isotypematched control antibodies demonstrating high specificity for theidiotype with no cross-reactivity to irrelevant Ig's.

Thus, the anti-idiotypic antibody 3H6 may be used, either directly as avaccine to induce protective and neutralizing immune responses, or as atool contributing to the design of an HIV-1 vaccine that alsosuccessfully induces protective and neutralizing B-cell responses. Ab2scan be suitable for vaccination of HIV-1 infected newborns, whose immunesystem might be tolerant to stimulation with other antigens [37, 38].

At the first glance Ab2 3H6 represents a vaccine antigen capable toinduce neutralizing immune responses. Immunization with Ab2 3H6 shallavoid the induction of antibody-dependent enhancement of infectionresponses (ADE) observed in polyclonal sera of HIV-1 positiveindividuals [39-42]. The Ab1 2F5 was passively administered ingram-quantities to human volunteers. No signs of ADE have been observedin these human volunteers despite the fact that complement activationwas detected [29]. As Ab2 3H6 induces 2F5-like specificities in the seraof immunized animals it can be expected that ADE-phenomena observed withpolyclonal sera of HIV-positive individuals [43] can be avoided uponvaccination with Ab2 3H6.

For the use as a pharmaceutical the antibodies and antibody fragments ofthe present invention may be part of a composition together with apharmaceutically acceptable carrier. Thus, in a further embodiment thepresent invention relates to pharmaceutical compositions, particularlyvaccines, which comprise the Ab2 antibodies or antibody fragments of thepresent invention together with a suitable, optionally immunogenic,carrier and/or adjuvant.

In yet another embodiment the present invention relates to Ab3antibodies (anti-anti-idiotypic antibodies) elicted by the present Ab2antibodies or antibody fragments of the 3H6 type, as well as topharmaceutical compositions, including vaccines, which comprise Ab3antibodies or antibody fragments directed against Ab2 3H6 and preferablydirected against the binding epitope of Ab2 3H6. In a most preferredembodiment, these Ab3 ntibodies or antibody fragments are of a naturesuch that they neutralize HIV-1. Pharmaceutical compositions containingsuch an Ab3 antibody or fragment thereof are useful for passiveimmunization against HIV-1.

The antibodies or antibody fragments of the present invention may beadministered in dosages of about 0.5 to about 10 μg/kg body weight, andpreferably boosted in one or more, usually periodic, intervals. Thepharmaceutical compositions of the present invention containing eitherAb2 3H6 antibodies or fragments thereof, or anti-3H6 Ab3 antibodies orfragments whereof may be administered prophylactically to prevent anindividual from getting HIV-1 infected, as well as therapeutically toinhibit or stop progression of disease in an HIV-1 infected individual.

EXAMPLE 1 Generation of 3H6 Antibodies

Animals:

Female BALB/c mice, 8-10 weeks old, were obtained from Charles River(Sulzfeld, Germany).

Antibodies:

Immunoglobulin subclass standards and isotype-specific anti-mouseantisera were purchased from Sigma (St. Louis, Mass.). Monoclonalanti-HIV-1 antibody 2F5 was purified and used as antigen bindingfragment (Fab′) for immunization. Antibodies were digested withMercuripapain (Sigma) and the digest purified with Protein-A-Sepharose 4FF-column.

Immunization of Animals:

50 μg of the 2F5 Fab′ were injected intraperetoneal once in completeFreund's adjuvant (CFA) and after three and six weeks again withincomplete Freund's adjuvant (IFA). Four days before spleen-ectomizationthe animals were boosted for the last time. Spleenocytes were preparedon day 46 after the first immunization.

Generation of Hybridoma Secreting Mouse IgG:

The serum titer of mice was used as criteria for the development ofimmunocompetent spleenocytes. Only mice with an anti-2F5 titer of atleast 1:100,000 were used for fusion. According to standard protocolsthe spleen cells were fused with X63-Ag-8.653 cells and plated in96-well plates. HAT-selection was started the day after fusion andsupernatants of growing wells were first tested for mouse IgG productionusing a mouse mAb as standard protein. Polyclonal goatanti-mouse-γ-chain antibody (Sigma) was diluted 1:500 in coating buffer(0.1N Na₂CO₃/NaHCO₃ pH 9.6) and 100 μL per well were precoated ontomicrotiter plates (ELISA grade I; Nunc, Denmark). After each incubationstep the plates were washed three times with PBS containing 0.1% Tween20 adjusted to pH 7.2. The standard mouse mAb was serially diluted,starting with 200 ng/mL (dilution buffer contained 1% BSA in washingbuffer). 50 μL per well of standard or serially diluted sample wereallowed to bind to the precoated well for 1 h, and after washing theplates, we used 50 μL of 1:5,000 diluted goat anti-mouse IgG+IgM polyHRP80 (RDI Inc.) as second antibody. After 1 h incubation time, theplates were stained with OPD and read at 492 nm (reference wavelength620 nm).

For the screening of antibodies against the 2F5 idiotype, thesupernatants of 2170 HAT-resistant Hybridoma were tested for IgGproduction. 65 positive clones were found. Approx. 10% of supernatantsof the IgG positive clones were also reacting with antibody 2F5.

EXAMPLE 2 Identification of Neutralizing Antibodies

Idiotype Binding Assay:

For initial screening of idiotype binding antibodies, 96-well microtiterplates were coated overnight at 4° C. with 2F5-IgG1, 2F5-IgG3 or 2G12,another gp120 recognizing human anti-HIV-1 antibody [31]. Samples werediluted 2⁸ in dilution buffer starting with 200 ng/mL. The Ab2s werevisualized as described for the mouse IgG ELISA.

The antibodies 3H6, 4F6, and 6F8 could be stabilized and were tested fortheir binding capacity in ELISA with different precoatings. Theanti-idiotypic antibody 1G1, directed against a different neutralizinganti-HIV-1 antibody, namely 2G12 (ECACC Acc. No. 93091517; for detailssee WO 96/33219), served as control. Table 1 shows the relative bindingproperties of Ab2s with different idiotypes. 4F6 and 6F8 reacted withall antibodies and seemed to recognize an epitope at the constantregion. The antibody 1G1 specifically binds to 2G12, while 3H6recognizes both isotypes of 2F5 and it was negative for 2G12.

TABLE 1 Ab2 2F5 IgG1 2F5 IgG3 2G12 IgG1 4F6 +++ ++ ++++ 6F8 ++ ++++ ++3H6 ++++ +++ − 1G1 − − +++Idiotype Inhibition Assay of Ab2 3H6:

Competitive binding assays were performed as previously described [32].Different concentrations of Ab2 (50, 500 ng/mL) were preincubated with1:2 dilutions of 2F5 starting with 100 ng/mL. After 1 hour theantibodies were allowed to react with the antigen on the plate. Thesolid phase was coated with the synthetic 2F5 epitope GGGLELDKWASL (SEQ.ID NO.13) at a concentration of 2 μg/mL and the final binding of 2F5 wasdetected as described [33].

The ELISA competition assay was designated to describe the remainingbinding capacity of 2F5 to the ELDKWA-epitope (SEQ ID NO: 1) afterpreincubation with Ab2. Ab2 3H6 inhibits or prevents the binding of 2F5to the epitope depending on the concentrations applied. FIG. 1 describesthe decline of 2F5 binding to the GGGLELDKWASL (SEQ ID NO. 13) precoatedplate with increasing concentrations of 3H6. Even 50 ng/mL 3H6 couldreduce the binding properties of 31 ng/mL 2F5 by more than one third(37% reduction of the OD) and 500 ng/mL resulted in 83% reduction of ODwhile the Ab2 6F8 did not diminish the binding of 2F5 to the epitope.Since the binding affinity of 2F5 to the GST- (glutathion S-transferase)ELDKWA (SEQ ID NO: 1) fusion-protein is 1.7×10⁷ M⁻¹ [33] the inhibitionpattern of Ab2 3H6 was acceptable for further studies.

Similar results were obtained when replacing GGGLELDKWASL (SEQ ID NO:13) with other synthetic peptides containing the ELDKWA (SEQ ID NO: 1)epitope or slightly modified, particularly functionally equivalent,variants thereof including functionally equivalent homologues orfunctionally equivalent variants occurring due to the degeneracy of thegenetic code and/or due to variability of the HIV-1 viruses, thevariants preferably being selected from the group consisting of ELDNWA(SEQ ID NO. 2), ELNKWA (SEQ ID NO. 3), LELDKWA (SEQ ID NO. 4), LELDNWA(SEQ ID NO. 5), LELNKWA (SEQ ID NO. 6), ELDKWAS (SEQ ID NO. 7), ELDNWAS(SEQ ID NO. 8), ELKNWAS (SEQ ID NO. 9), LELDKWAS (SEQ ID NO. 10),LELDNWAS (SEQ ID NO. 11), LELNKWAS (SEQ ID NO. 12).

Virus Neutralization Assay with 3H6:

Interaction of 2F5 with the different Ab2s was further investigated in aneutralization assay as described previously [26]. The assay wasperformed with HIV-1 strain NL4-3 infected AA-2 cells (NIAID, Bethesda,Md.) as indicator cells using p24 antigen as virus replication marker.Serial dilutions of Ab2 antibodies (starting concentration of 250 μg/mL)were mixed with constant amounts of 2F5 or 2G12 as negative control (10μg/mL of each neutralizing antibody) and pre-incubation with virusbefore addition of AA-2 cells and further incubated for 5 days. Virusreplication was measured in a p24-ELISA at the time point of terminationof the assay [34]. The ratios of p24 antigen production in Ab-containingcultures (Vn) to p24 antigen production in control cultures (Vo), takinginto account the input p24, were calculated and Ab concentrations(μg/mL) causing x % neutralization were plotted. Each test included avirus titration to determine the actual tissue culture infectious dose50 (TCID₅₀) and the viral titer was determined by linear regressionanalysis. Tests were considered to be valid in case of viral titersbetween 10²-10³ TCID₅₀.

FIG. 2 shows the in vitro neutralization of HIV-1 NL4-3 afterpreincubation of Ab2 3H6 with 2F5 and 2G12. 2F5 and 2G12 aloneneutralize 90% of the virus at a concentration of 3 μg/mL and 99% of thevirus at 12 μg/mL and 11 μg/mL, respectively (data not shown). The 2F5and 2G12 concentration was kept constant at 10 μg/mL. 50 μL of 2F5 or2G12 were incubated with serial dilution of the Ab2s, 3H6, 4F6, 6F8 andonly 3H6 was able to deplete neutralization of 2F5 (4F6 and 6F8 are notplotted). FIG. 1 b describes the percent neutralization representing theremaining neutralizing capacity of 10 μg/mL 2F5 and 2G12 with increasingamounts of 3H6. 3.9 μg/mL of 3H6 extinguish the 2F5 neutralizationproperties and 1.9 μg/mL are able to reduce neutralizing capacity of 2F5for 5%. This means that 3H6 and 2F5 react in nearly equimolar ratio,since the molecular weight of the two corresponding antibodies may varydue to oligomerization or cleavage. 3H6 is not able to reduceneutralization of 2G12 at concentration as high as 250 μg/mL. Ab2 3H6was able to reduce 2F5 neutralization for 95% at 3.9 μg/mL.

Sequence of the variable regions of the heavy and light chains ofantibody 3H6:

-   a) Amino acid sequence of the heavy chain variable region (vH) of    anti-idiotypic antibody 3H6 (SEQ ID NO. 14):

GVQLQQSGPELVKTGASVKISCKASGYSFTDYFMHWVKQSHGKSLDWIGYINCYTGATNYSQKFKGKATFTVDTSSNTAYMQFNSLTSEDSAVYYCARTSIGYGSSPPFPYWGQGTLVTVSA

-   b) Amino acid sequence of the light chain variable region (vL) of    anti-idiotypic antibody 3H6 (SEQ ID NO. 15):

ETTVTQSPASLSMSIGEKVTIRCITSTDIDDDMNWYQQKPGEPPRLLISDGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGG GTNLEIK

EXAMPLE 3 Induction of Anti-Anti-Idiotypic Antibodies (Ab3)

Induction of Ab3 in BALB/c Mice:

After purification of Ab2 (3H6) by affinity chromatography it wasdigested with Mercuripapain (Sigma) and the digest purified withProtein-A-Sepharose 4 FF-column. Three BALB/c mice were injected with 5μg of the 3H6Fab′ intraperetoneally and after three weeks again, bothtimes with Freund's incomplete adjuvant. Another week later the micewere sacrificed and blood and ascites samples were collected. Thesamples were analyzed by ELISA for IgG titer as described above.

Three BALB/c mice were immunized with 5 μg/mL 3H6Fab′ fragment FIAsolution. Unfortunately, after the second immunization the animalsdeveloped ascites and had to be sacrificed without finishing theimmunization scheme. Serum and ascites were shown to have total IgGtiters up to 2 mg/ml. The results of IgG titers, binding capacity andcompetition assay are summarized in Table 2. The binding to GGGLELDKWASL(SEQ. ID NO.13) and gp160 in an ELISA was illustrated in FIG. 2 byplotting the cut-off dilution of the polyclonal serum and ascites. Endpoint titers (cut-off) were calculated as the reciprocal of the highestdilution that resulted in an OD at least two times greater than the ODobtained with corresponding preimmune IgG.

TABLE 2 Cut-off Cut-off 50% inhi- titer titer bition of Neu- IgG bindingbinding to 2F5/ tralizing titer to ELDKWA gp 160 capacity Sample [μg/mL]gp 160 (SEQ ID NO: 1) binding (IC₅₀) Serum 1 2271 1280 1280 1:60 — Serum2 3776 1280 1280  1:120 1:14 Serum 3 2603 1280 1280  1:120 1:12 Ascites1 1127 320 320 1:15 — Ascites 2 1958 640 640 1:15 — Ascites 3 1442 320320 1:15 —Characterization of Ab3:Specificity of Ab3:

Serum and ascites samples of the 3H6 immunized mice were assayed forbinding to the synthetic epitope GGGLELDKWASL (SEQ. ID NO.13) (1 μg/mLin coating buffer) and recombinant gp160 (1 μg/mL in coating buffer).The mouse antibodies were detected as described above. In anotherspecificity test the serial dilutions of Ab3 samples were coincubatedwith a constant amount of 2F5 on a gp160 precoated plate. The 2F5binding capacity at increasing dilutions of Ab3, starting with 1:10, wasexamined.

The 2F5 antibody was utilized at 25 ng/mL and a serial dilution of Ab3samples starting with 1:10 was used as competitor. Ab3 sera that mostpotently competed with 2F5 binding on the antigen also exhibited invitro neutralization potency. FIG. 3 illustrates the reduction of 2F5binding to the precoated gp160 in terms of competition with the Ab3samples.

Syncitium Inhibition Assay:

The neutralizing activity of Ab3 sera and ascites was assessed byinhibition of syncitium formation of HIV-1 strain RF_(NT). Serialdilutions of serum, ascites or control antibody (2F5) were pre-incubatedwith virus (10²-10³ TCID₅₀/ml) for 1 h at 37° C. before addition of AA-2cells. Starting dilutions of serum and ascites were 1:5 in culturemedium. Cells were incubated for 5 days before assessment of syncitiumformation. Experiments were performed with 4 replicates per dilutionstep. The presence of at least one syncitium per well was considered asindication for HIV-1 infection. The 50% inhibiting concentration (IC₅₀)was calculated according to the method of Reed and Muench [35].Experiments included a virus titration of the inoculum to confirm theinfectious titer.

The neutralizing properties of serum and ascites were tested with HIV-1laboratory strain RF_(NT). Samples were diluted initially 1:5,sterilized by 0.2μ filtration and used at a starting concentration of1:10 in the AA-2 syncytia inhibition assay in quadruplicates. Thevirus-titer (TCID₅₀/ml) was determinated to be 10^(2.75). The ascitessamples and serum 1 did not show any antiviral effect. Sera 2 and 3reached an IC50 at dilutions 1:14.1 and 1:11.9 respectively. Serum 1didn't show neutralization as well as the ascites samples.

EXAMPLE 4 Cytokine-Associated Antibody

3H6 vL and vH coding sequences are isolated from the murine mAb Ab2/3H6via RT-PCR from the cultivated hybridoma cell line (see Example 1).

Thereafter human Cκ and IgG Fc domains are fused to the variablefragments of 3H6 by SOE-PCR (splicing by overlapping extension) andsubsequently cloned into the eucaryotic expression vector pIRES. Thiscloning step results in a bicistronic expression cassette withCMV-promoter—chimeric 3H6 heavy chain-internal ribosomal entry site(IRES)—chimeric 3H6 light chain—SV40 polyA. The final plasmid is calledp3H6 mhHC/LC.

Human IL-15 cDNA is amplified by PCR and fused to the antibody heavychain domain by SOE-PCR resulting in a fusion protein mo/hu3H6/IL-15 asshown in FIG. 6. This mo/hu3H6/IL-15 heavy chain replaces the 3H6chimeric heavy chain in the expression plasmid p3H6 mhHC/LC resulting inthe second expression plasmid p3H6 mhHC-IL-15/LC.

Two recombinant CHO-cell-lines are generated by cotransfection: one cellline expressing the mouse/human chimeric antibody mo/hu3H6 and thesecond cell-line expressing the same antibody with human IL-15 at theC-terminus of the 3H6 heavy chain (mo/hu3H6/IL-15).

The target plasmids described above are cotransfected together with theplasmid pdhfr coding for the mouse dihydrofolate-reductase (dhfr) byLipofectin®. Subsequent selection of transfected cells is performedusing geneticin sulfate (G418). Methotrexate (MTX) is used asamplification marker. After expansion of the subclones, screening forthe best producing clone is done with an ELISA and flow cytometry with acommercial anti-human IL-15 mAb and the mAb 2F5. The selection processis repeated before the subclone with the highest secretion rate of thedesired protein is expanded.

Thereafter, quantification and comparison of the amounts of secreted andintracellularly retained protein of the mo/hu3H6 or mo/hu3H6/IL-15fusion protein is carried out.

Protein purification is done by affinity chromatography, optionallyfollowed by ion-exchange chromatography. The isolated and purifed fusionprotein mo/hu3H6/IL-15 is subjected to analytical determination of thebiochemical properties including determination of:

-   -   affinity to the IL-2/IL-15-R using the Biacore or Isothermal        Microcalorimetry;    -   molecule size using gel filtration columns and native gel        electrophoresis;    -   in vitro stability of the immunocytokine by incubation in human        serum;    -   competition assay of the mo/hu3H6/IL-15 fusion protein with the        mo/hu3H6 antibody molecule to compare their binding capacity;    -   In vitro neutralisation experiments to describe the inhibiting        properties of the mo/hu3H6 and the mo/hu3H6/IL-15 fusion        proteins    -   Diffusion through biological membranes, binding to the        corresponding receptors, e.g. IL-2/IL-15-R on selected tissues        using immuno-histological assays.

The construction and expression of the mo/hu3H6/IL-4 fusion protein isdone analogously.

The results from in vitro experiments confirm both the HIV-1neutralisation activity as well as the B cell response stimulatingactivity of the above disclosed fusion protein constructs of the presentinvention (data not shown herein).

REFERENCES

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1. A monoclonal antibody or fragment thereof which is reactive withHIV-1 neutralizing antibody 2F5 produced by a hybridoma cell line havingthe ECACC Accession No. 90091704 and which inhibits or prevents eitheror both of the HIV- 1 neutralization activity of antibody 2F5 or thebinding of antibody 2F5 to gp4l of I-IIV-1, the fragment being anantigen-binding part of the antibody which retains the bindingspecificity of the whole antibody, wherein the antibody isanti-idiotypic to antibody 2F5 and is produced by hybridoma cell line3H6 having the ECACC Acc. No.
 01100279. 2. The antibody or fragmentthereof according to claim 1, wherein it inhibits or prevents binding ofantibody 2F5 to proteins or peptides that comprise an amino acidsequence selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO12.
 3. The antibody or fragment thereof according to claim 1, wherein itelicits upon administration to a mammal Ab3-type antibodies that haveeither or both of an HIV-1 neutralizing activity, or an ability tocompete with antibody 2F5 for binding to proteins or peptides thatcomprise an amino acid sequence selected from the group consisting ofSEQ ID NO. 1 SEQ ID NO
 12. 4. The antibody or fragment thereof accordingto claim 1, wherein the antibody has either or both of a heavy chainvariable region according to SEQ ID NO 14 or light chain variable regionaccording to SEQ ID NO
 15. 5. The antibody or fragment thereof accordingto claim 1, wherein the antibody is a chimeric mouse/human antibody or ahumanized version of a mouse antibody.
 6. The antibody or fragmentthereof according to claim 1, wherein it is linked to an immunoactivemolecule that increases or intensifies a B cell response.
 7. A hybridomacell line producing an antibody according to claim 1, the cell linebeing 3H6 deposited at PHLS, Porton Down, Salisbury, UK, under ECACCAcc. No.
 01100279. 8. A composition comprising an antibody or antibodyfragment defined in claim 1, and a pharmaceutically acceptable carrier.9. The antibody or fragment thereof according to claim 6, wherein theimmunoactive molecule is a cytokine selected from the group consistingof interleukin-4 (IL-4) and interleukin-1 5 (IL-15).